1. Introduction

The World Health Organization estimated that 296 million people were infected with chronic hepatitis B, which resulted in 820,000 deaths, mostly from cirrhosis and hepatocellular carcinoma (HCC) in 2019.[1] The genome of hepatitis B virus (HBV) translocates to the nucleus and a covalently closed circular DNA (cccDNA) is formed after HBV infects hepatocytes.[2] It is difficult to eliminate HBV completely because of the stability of cccDNA. The level of cccDNA in the liver correlates with the serum level of hepatitis B surface antigen (HBsAg),[3] and it has been reported that high levels of HBsAg increase the risk of liver carcinogenesis in patients with a low viral load.[4] The goal of anti-HBV therapies is the removal of serum HBsAg, termed “functional cure,”[5,6] but this is rarely achieved with clinically available therapies such as nucleos(t)ide analogues (NAs) and interferons (IFNs). HBsAg quantification is considered to be a surrogate marker of efficient viral suppression during NA treatments that make serum HBV DNA undetectable in most patients.[7]

NAs including entecavir (ETV) and tenofovir disoproxil fumarate (TDF) have been widely used for the treatment of chronic HBV infection.[8] Both agents inhibit the reverse transcription of the HBV genome and HBV DNA in the serum can be reduced rapidly. Therefore, these have been considered a first-line therapy. However, ETV has minimal efficacy in reducing the serum HBsAg levels and in reducing the risk of developing of HCC in chronic hepatitis B patients with advanced liver fibrosis.[9–11] Also, long-term use of TDF causes renal toxic effects in some patients and is associated with a reduction in mineral bone density and an increase in markers of bone turnover.[12,13]

A novel NA, tenofovir alafenamide fumarate (TAF), has also been available from 2017 in Japan for the treatment of chronic hepatitis B. TAF is a new prodrug of tenofovir that is as effective as TDF in HBV suppression. Also, because TAF has greater stability in plasma than TDF, TAF is able to deliver the active metabolite more efficiently to target cells at a substantially lower dose.[12,13] For the above reasons, TAF was added to the first-line antiviral agents for the treatment of HBV infection.[5,6]

It was reported that TDF reduces the serum HBsAg level significantly more than ETV[10] and that TAF is not inferior to TDF in HBsAg suppression.[12,13] Therefore, it is assumed that the efficacy of TAF in HBsAg reduction may be superior to ETV. ETV has been widely used in Japan since 2006, and some studies have reported the efficacies of treatment switch from ETV to TAF. However, there has been no randomized study for the evaluation of ETV-TAF switch so far and it is still questioned whether such a treatment switch is better than continuation of ETV. In this study, we firstly performed a prospective randomized controlled study to verify the hypothesis that the HBsAg reduction in the TAF-switching group may be significantly greater than that in the ETV-continuing group.

2. Materials and Methods 2.1. Study design, setting, and sample sizes

This study was a prospective open-label multicenter randomized double-arm controlled trial. The participants were assigned to a TAF-switching group or an ETV-continuing group equally (Fig. 1) using a random number table created by a random number program at the research office, which were not disclosed to investigators. Based on the average decrease in HBsAg 48 weeks after administration in a phase 3 clinical study for TDF, it was calculated that 52 patients in each group were required to show a significant difference under a condition of an α value 0.05 in the two-sided test and an analysis power 0.8. With the expectation of dropout of about 10%, the inclusion of 60 patients in each group was planned.

Figure 1.:

Flow-chart of randomization and evaluation in this study.

Inclusion criteria were as follows: ETV 0.5 mg/day had been administered for >1 year continuously; HBsAg in the serum had been continuously positive; The serum HBV DNA levels were less than 3.3 log IU/mL; patients ≥ 20 years old; they had no history of decompensated liver cirrhosis, liver cancer, or other malignancies. The exclusion criteria were as follows: patients receiving IFN or immunosuppressive therapies; presence of hypophosphatemia (<2.5 mg/dL); pregnant women and women suspected of being pregnant; breast-feeding women; and coinfection with hepatitis C virus or human immunodeficiency virus.

2.2. Participants

Participants were enrolled from April 2018 to June 2019 and were observed for 2 years until March 2021 in 5 hospitals. Among the 33 enrolled patients, 3 patients were excluded due to unscheduled treatments and a total of 30 patients were evaluated. The median age of the randomized patients was 61 years old (interquartile range, 50–68); 18 patients (60%) were male, and 12 patients (40%) were female. After randomization, 16 patients (median age, 63; interquartile range, 50–68; 7 males and 9 females) were evaluated as the TAF switching group and 14 patients (median age, 60; interquartile range, 51–68; 11 males and 3 females) were evaluated as the ETV continuing group.

2.3. Intervention

ETV at 0.5 mg/day was administered orally while fasting, and TAF at 25 mg/day was administered orally after a meal. The patients were observed every 3 months for 24 months and the clinical data were collected at 3, 6, 9, 12, 18, and 24 months after enrollment.

2.4. Comparison

The serum levels of HBsAg were quantified using a chemiluminescent enzyme immunoassay with LUMIPULSE HBsAg-HQ (Fujirebio, Tokyo, Japan). Hepatitis B e antigen (HBeAg) was assessed using a chemiluminescent immunoassay by ARCHITECT (Abbott Japan, Tokyo, Japan). The HBV DNA levels were quantified using quantitative PCR assays with Cobas TaqMan HBV Auto, according to the manufacturer’s protocol (Roche Diagnostics, Tokyo, Japan). Hepatitis B core-related antigen (HBcrAg) was tested using a chemiluminescent enzyme immunoassay with LUMIPULSE (Fujirebio). HBV genotypes were determined using the IMMUNIS HBV genotype EIA kit (Institute of Immunology, Tokyo, Japan). As a liver fibrosis marker, FIB-4 index was calculated as follows: FIB-4 = age (years) × aspartate aminotransferase (U/L)/(platelet counts [PLT, 109/l] × √ alanine aminotransferase [ALT, U/L]).[14] Imaging tests including abdominal ultrasonography or computed tomography test were performed for the screening of liver cancer.

2.5. Ethics and end point

The primary efficacy endpoint was the change of serum HBsAg and HBsAg normalization at 24 months, and the secondary endpoints were the serum HBV DNA sustained normalization, the changes of ALT, the HBeAg sero-clearance, estimated glomerular filtration rate (eGFR), and inorganic phosphorus (IP).

The description of the Case Report Form for the data collection was made by the attending physicians. The principal investigators of each institution submitted the case report form to the research office. Before the submission, information that could identify an individual was deleted. This study was registered on University Hospital Medical Information Network Clinical Trials Registry (UMIN-CTR, ID: UMIN000032201). The study protocol conformed to the guidelines described in the Declaration of Helsinki and was approved by the Medical Ethics Committee of Tohoku University (approval no. 2018-2-177). Written informed consent was obtained from each patient.

2.6. Statistical analysis

Statistical analysis was performed using JMP version 14.2 (SAS Institute Inc., Cary, NC). Statistical comparisons were performed using a χ2 test for the comparison of frequencies between the 2 groups or a Wilcoxon rank sum test for the comparison of continuous variables between 2 groups. As an additional analysis to eliminate the effects of an uneven distribution of gender, only male patients were analyzed using analysis of covariance (ANCOVA) to adjust the parameters with baseline data in comparisons between the TAF and ETV groups. P < .05 was considered to indicate a statistically significant difference.

3. Results 3.1. Clinical characteristics of the enrolled patients

The mean age of the 30 patients who were evaluated in the present study was 61 years old and 18 patients (60%) were male. The median ALT, PLT, and HBsAg were 19 U/L, 18.2 × 104/μL, and 2.9 log IU/mL, respectively. The HBV DNA of all patients were below detection limit. A total of 11 (37%) and 18 (60%) patients were infected with HBV genotype B and C, respectively, and 1 patient was undetermined. The clinical characteristics of the chronic hepatitis B patients in the TAF switching group (n = 16) and the ETV continuing group (n = 14) are shown in Table 1. The proportion of males and the serum levels of γ-GTP in the TAF group were significantly higher than those in the ETV group. Other characteristics were not significantly different between the 2 groups. The serum HBV DNA levels were undetectable in all patients.

Table 1 - Clinical characteristics of the patients evaluated in this study. Characteristics Overall, n = 30 TAF group, n = 16 ETV group, n = 14 P value (TAF vs ETV) Age, yr 61 (39–75) 63 (43–74) 60 (39–75) .819 Sex, male/female 18/12 7/9 11/3 .048 T-Bil, mg/dL 0.9 (0.7–1.1) 0.8 (0.7–1.0) 0.9 (0.8–1.2) .346 AST, U/L 23 (21–27) 22 (19–26) 23 (21–27) .358 ALT, U/L 19 (17–24) 18 (17–21) 24 (17–39) .054 γ-GTP, U/L 19 (17–40) 18 (17–19) 26 (19–47) .009 Alb, g/dL 4.4 (4.2–4.6) 4.3 (4.2–4.5) 4.5 (4.4–4.7) .200 Cr, mg/dL 0.77 (0.66–0.91) 0.75 (0.62–0.86) 0.83 (0.69–1.00) .114 eGFR, mL/min/1.73 m2 59.2 (49.5–72.4) 61.0 (53.4–77.0) 55.1 (47.6–69.0) .146 IP, mg/dL 3.3 (3.1–3.7) 3.2 (3.1–3.6) 3.5 (3.3–3.6) .499 PLT, ×104/μL 18.2 (15.5–22.9) 17.8 (15.5–22.9) 18.7 (16.1–21.1) .983 FIB-4 index 1.59 (1.17–2.17) 1.62 (1.26–2.14) 1.55 (1.13–2.10) .819 AFP, ng/mL 2.9 (2.2–3.4) 2.6 (2.1–3.4) 2.9 (2.2–3.3) 1.000 HBV DNA, log IU/mL BDL (BDL-BDL) BDL (BDL-BDL) BDL (BDL-BDL) 1.000 HBsAg, log IU/mL 2.90 (2.38–3.16) 2.86 (2.38–3.32) 2.93 (2.67–3.06) .575 HBeAg, +/– 3/27 4/12 2/12 .507 HBcrAg, log U/mL 3.6 (3.2–5.3) 3.2 (BDL-3.4) BDL (BDL-1.9) .608 HBV genotype, B/C/unknown 11/18/1 6/9/1 5/9/0 .511 Prior ETV duration, months 81 (43–105) 87 (49–119) 59 (42–89) .329 Previous IFN-α treatment, yes/no 1/29 1/15 0/14 .340

Median (interquartile range) or number is indicated.

AFP = α fetoprotein, Alb = albumin, ALT = alanine aminotransferase, AST = aspartate aminotransferase, BDL = below detection limit, Cr = creatinine, eGFR = estimated glomerular filtration rate, ETV = entecavir, γ-GTP = γ-glutamyltransferase, HBcrAg = hepatitis B core-related antigen, HBeAg = hepatitis B e antigen, HBsAg = hepatitis B surface antigen, HBV = hepatitis B virus, IP = inorganic phosphorus, PLT = platelet counts, TAF = tenofovir arafenamide, T-Bil = total bilirubin.

3.2. Comparison of the antiviral effects between the TAF and ETV groups

At first, the change of HBsAg from the randomization (ΔHBsAg) was evaluated. A minus value indicates a decline of HBsAg in comparison with the baseline. There was no significant difference in the mean change of HBsAg at 12 months (–0.06 vs –0.11 log IU/mL, P = .17) and at 24 months (–0.08 vs –0.20 log IU/mL, P = .07) between the TAF and ETV groups (Fig. 2A).

Figure 2.:

Comparison of the changes in the assessed parameters from baseline between TAF-switching group and ETV-continuing group. (A–D) The changes of HBsAg (A), ALT (B), eGFR (C), and IP (D). (E,F) eGFR change in patients with eGFR ≥ 60 mL/min/1.73 m2 (E) and eGFR < 60 mL/min/1.73 m2 (F). Solid lines indicate means of TAF-switching group and dotted lines indicate those of ETV-continuing group. Error bars indicate standard deviations (SDs). *, P < .05. ALT = alanine aminotransferase, eGFR = estimated glomerular filtration rate, ETV = entecavir, HBsAg = hepatitis B surface antigen, IP = inorganic phosphorus, TAF = tenofovir alafenamide fumarate.

The mean changes in ALT (ΔALT) from the baseline were similar to those of HBsAg in both groups at 12 months (0.4 vs –2.4 U/l, P = .08) and at 24 months (0.5 vs –1.0 U/l, P = .17) (Fig. 2B). There were 3 patients who were positive for HBeAg at randomization, and HBeAg sero-clearance was achieved in 1/2 (50%) patients in the TAF group and 0/1 (0%) patients in the ETV group. No patients developed liver cancer during the observation period.

At the time of randomization, the proportion of males in the ETV group was significantly higher than that in the TAF group. Then, to eliminate the effect of uneven distribution of gender, we analyzed only male patients (Table 2) and compared the parameters using ANCOVA to adjust with the baseline data (Fig. 3). ΔHBsAg was not significantly different between the TAF group and the ETV group at 24 months (–0.14 vs –0.22 log IU/mL, P = .57) (Fig. 3A). Similarly, ΔALT was not significantly different between the two groups (–4.5 vs 0.3 U/L, P = .83) (Fig. 3B).

Table 2 - Clinical characteristics of the male patients evaluated in this study. Characteristics Overall, n = 18 TAF group, n = 7 ETV group, n = 11 P value (TAF vs ETV) Age, yr 61 (49–68) 64 (51–67) 59 (47–68) .751 T-Bil, mg/dL 1.0 (0.9–1.2) 1.0 (0.9–1.2) 0.9 (0.9–1.2) .584 AST, U/L 23 (21–27) 22 (20–27) 23 (21–31) .585 ALT, U/L 22 (17–35) 20 (18–24) 24 (18–40) .275 γ-GTP, U/L 24 (18–44) 17 (16–35) 27 (20–55) .160 Alb, g/dL 4.5 (4.2–4.6) 4.2 (4.1–4.5) 4.5 (4.4–4.8) .158 Cr, mg/dL 0.86 (0.79–0.94) 0.86 (0.82–0.89) 0.92 (0.79–0.96) .555 eGFR, mL/min/1.73 m2 50.9 (47.8–57.8) 51.4 (50.1–56.1) 48.8 (45.5–61.2) .497 IP, mg/dL 3.1 (2.8–3.6) 2.8 (2.5–3.0) 3.5 (3.1–3.7) .059 PLT, ×104/mL 18.7 (16.2–21.5) 16.4 (15.2–19.8) 19.2 (18.2–22.4) .258 FIB-4 index 1.51 (1.13–2.00) 1.60 (1.37–2.21) 1.50 (1.05–1.60) .298 AFP, ng/mL 2.9 (2.2–3.5) 2.8 (2.4–3.3) 2.9 (2.1–3.5) .957 HBV DNA, log IU/mL BDL (BDL-BDL) BDL (BDL-BDL) BDL (BDL-BDL) 1.000 HBsAg, IU/mL 762 (231–1157) 241 (166–721) 835 (578–1247) .160 HBeAg, +/– 1/17 0/7 1/10 1.000 HBcrAg, log U/mL BDL (BDL-BDL) BDL (BDL-1.6) BDL (BDL-BDL) .317 HBV genotype, B/C/unknown 7/11/0 3/4/0 4/7/0 .783 Prior ETV duration, months 54 (41–101) 82 (41–111) 49 (41–87) .618 Previous IFN-α treatment, yes/no 0/18 0/7 0/11 -

Median (interquartile range) or number is indicated.

AFP = α fetoprotein, Alb = albumin, ALT = alanine aminotransferase, AST = aspartate aminotransferase, BDL = below detection limit, Cr = creatinine, eGFR = estimated glomerular filtration rate, ETV = entecavir, γ-GTP = γ-glutamyltransferase, HBcrAg = hepatitis B core-related antigen, HBeAg = hepatitis B e antigen, HBsAg = hepatitis B surface antigen, HBV = hepatitis B virus, IP = inorganic phosphorus, PLT = platelet counts, TAF = tenofovir arafenamide, T-Bil = total bilirubin.

Figure 3.:

Comparison of the changes in the assessed parameters from baseline between TAF-switching group and ETV-continuing group only in male patients. (A–D) The changes of HBsAg (A), ALT (B), eGFR (C), and IP (D). Solid lines indicate means of TAF-switching group and dotted lines indicate those of ETV-continuing group. Error bars indicate standard deviations (SDs). ALT = alanine aminotransferase, eGFR = estimated glomerular filtration rate, ETV = entecavir, HBsAg = hepatitis B surface antigen, IP = inorganic phosphorus, TAF = tenofovir alafenamide fumarate.

3.3. Factors associated with HBsAg decline

Table 3 shows the clinical baseline characteristics of patients with a HBsAg decline of 0.1 log IU/mL or more (HBsAg-declined group) and those with a HBsAg decline less than 0.1 log IU/mL (HBsAg-stable group) at 24 months in overall patients. Because 6 patients lacked data of HBsAg at 24 months, we evaluated 24 cases for this analysis. Interestingly, prior ETV duration was significantly shorter in the HBsAg-declined group than in the HBsAg-stable group (49 vs 92 months, P = .03). The levels of γ-GTP tended to be higher in the HBsAg-declined group than in the HBsAg-stable group (27 vs 18 U/l, P = .06). The reason is unclear, but because the γ-GTP levels were significantly higher in the ETV group at baseline, there was a possibility that the ETV group might had an advantage at this point due to the uneven distribution of gender. There was no statistical difference in FIB-4 index and HBsAg levels.

Table 3 - Clinical characteristics of the patients with HBsAg-declined group or HBsAg-stable group. Characteristics HBsAg-declined*, n = 13 HBsAg-stable†, n = 11 P value Age, yr 54 (42–74) 59 (43–74) .310 Sex, male/female 9/4 4/7 .107 T-Bil, mg/dL 0.9 (0.7–1.0) 0.8 (0.8–1.2) .537 AST, U/L 25 (20–31) 22 (21–23) .210 ALT, U/L 24 (15–40) 18 (17–22) .162 γ-GTP, U/L 27 (18–47) 18 (14–20) .055 Alb, g/dL 4.4 (4.1–4.6) 4.4 (4.3–4.6) .415 Cr, mg/dL 0.76 (0.67–0.89) 0.73 (0.62–0.96) .862 eGFR, mL/min/1.73 m2 61.9 (52.8–73.5) 60.3 (48.8–80.7) .931 IP, mg/dL 3.5 (3.1–3.8) 3.3 (3.0–3.7) .539 PLT, ×104/μL 17.7 (14.4–20.2) 20.9 (15.6–23.6) .385 FIB-4 index 1.50 (1.15–2.15) 1.53 (1.06–2.16) .931 AFP, ng/mL 2.8 (2.2–4.1) 2.9 (2.1–3.8) .843 HBV DNA, log IU/mL BDL (BDL-BDL) BDL (BDL-BDL) - HBsAg, log IU/mL 2.91 (2.56–3.17) 3.09 (2.38–3.45) .543 HBeAg, +/– 1/12 2/9 .439 HBcrAg, log U/mL 4.3 (3.7–4.8) 3.4 (3.2–5.8) .696 HBV genotype, B/C/unknown 5/8/0 3/7/1 .494 Prior ETV duration, months 49 (14–117) 92 (14–175) .030 Previous IFN-α treatment, yes/no 0/13 1/10 .267 Treatment group, TAF/ETV 5/8 7/4 .219

Median (interquartile range) or number is indicated.

AFP = α fetoprotein, Alb = albumin, ALT = alanine aminotransferase, AST = aspartate aminotransferase, BDL = below detection limit, Cr = creatinine, eGFR = estimated glomerular filtration rate, ETV = entecavir, γ-GTP = γ-glutamyltransferase, HBcrAg = hepatitis B core-related antigen, HBeAg = hepatitis B e antigen, HBsAg = hepatitis B surface antigen, HBV = hepatitis B virus, IP = inorganic phosphorus, PLT = platelet counts, TAF = tenofovir arafenamide, T-Bil = total bilirubin.

* ⊿ HBsAg (24 months) ≤ –0.1 logIU/mL.

† ⊿ HBsAg (24 months) > –0.1 logIU/mL.

Next, the clinical baseline characteristics were compared to patients with HBsAg decline of 0.1 log IU/mL or more and those without in the TAF group (Table 4). HBsAg-declined patients tended to have lower PLT (16.4 vs 22.7 × 104/μL, P = .09) and higher FIB-4 levels (2.08 vs 1.26, P = .12). Therefore, it was suggested that switching to TAF might decrease HBsAg more in patients with advanced liver fibrosis. Also, HBsAg-declined patients tended to have lower HBsAg (2.85 vs 3.34 log IU/mL, P = .17) at randomization. The patients with both low PLT (<19.3 × 104/μL) and low HBsAg (<3.46 log IU/mL) at randomization had a significantly larger decline in HBsAg than the patients without (–0.1 vs –0.05 log IU/mL, P = .03) (Fig. 4A). Figure 4B shows the HBsAg change in each patient in TAF-group with both low PLT and low HBsAg or those without. The HBsAg levels of all 8 patients with both low PLT and low HBsAg declined from baseline to 24 months. Importantly, such a difference was not observed in the ETV group (–0.16 vs –0.21 log IU/mL, P = .87). The patients with high FIB-4 index (≥1.67) and those with a short prior ETV duration (<87 months) tended to have a larger decline of HBsAg, but the differences were not significant (Fig. 4A).

Table 4 - Clinical characteristics of the patients with HBsAg-declined or HBsAg-stable in TAF-switching group. Characteristics TAF group HBsAg-declined*, n = 5 HBsAg-stable†, n = 7 P value Age, yr 53 (47–68) 59 (46–66) .935 Sex, male/female 3/2 1/6 .098 T-Bil, mg/dL 0.8 (0.7–1.2) 0.8 (0.6–0.8) .671 AST, U/L 26 (18–27) 22 (21–23) .566 ALT, U/L 18 (13–26) 18 (17–19) .935 γ-GTP, U/L 18 (16–45) 17 (13–18) .190 Alb, g/dL 4.4 (4.1–4.7) 4.3 (4.2–4.4) .849 Cr, mg/dL 0.78 (0.66–0.90) 0.69 (0.59–0.76) .168 eGFR, mL/min/1.73 m2 61.6 (52.8–68.8) 69.6 (58.2–82.2) .223 IP, mg/dL 3.3 (3.0–3.9) 3.2 (3.1–3.8) .924 PLT, ×104/μL 16.4 (14.2–17.8) 22.7 (15.6–24.9) .088 FIB-4 index 2.08 (1.37–2.27) 1.26 (1.06–2.13) .123 AFP, ng/mL 2.4 (2.2–4.6) 2.9 (1.7–5.1) .705 HBV DNA, log IU/mL BDL (BDL-BDL) BDL (BDL-BDL) 1.000 HBsAg, log IU/mL 2.85 (2.37–3.15) 3.34 (2.75–3.62) .168 HBeAg, +/– 0/5 2/5 .190 HBcrAg, log U/mL BDL (BDL-BDL) 3.4 (3.2–5.8) - HBV genotype, B/C/unknown 3/2/0 2/4/1 .455 Prior ETV duration, months 48.8 (33.6–98.7) 92.4 (78.0–124.9) .223 Previous IFN-α treatment, yes/no 0/5 1/6 .377

Median (interquartile range)